Analysis of the constituents in jojoba wax used as a food additive by LC/MS/MS

Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.     

Analysis of rubusoside and related compounds in tenryocha extract sweetener

The main and minor constituents of tenryocha extract product, a natural sweetener, were investigated as a part of an ongoing study to evaluate its quality and safety as a food additive. Four constituents, namely rubusoside, steviolmonoside, panicloside IV, and ent-16 alpha, 17-dihydroxykauran-19-oic acid, were isolated. The concentration of rubusoside, the main sweet constituent, was 8.65% in the tenryocha extract product. In addition, it was confirmed that the origin of the extract was the leaves of Rubus suavissimus S. Lee (Rosaseae), as determined by comparing TLC and HPLC profiles of the product and hot-water extract prepared from the leaves of R. suavissimus.     

Cell culture in a closed nano-space

The minimum size of a closed nano-space in which cells can survive was determined using 4-nl nanowells. One or two cells could divide in the nanowell. Our results suggest that the cell division activity in the nano-space is determined by the conflict between intercellular effects and consumption of substrates.     

Simultaneous determination of aconitine analogues in Aconitum plants and foods that caused food poisoning by liquid chromatography with tandem mass spectrometry

A simple method for the simultaneous determination of four aconitine analogues (AC; aconitine, HA; hypaconitine, MA; mesaconitine, JA; jesaconitine) in Aconitum plants (Aconitum subcuneatum NAKAI) and a food that caused food poisoning was developed, using liquid chromatography tandem mass spectrometry (LC/MS/MS). Aconitine analogues were extracted with 1 mmol/L HCl and then cleaned up with an Oasis HLB cartridge. The LC separation was performed with an octadecylated silica column (Develosil ODS-HG-5, 2.0 mm i.d. x 50 mm) at a flow rate of 0.2 mL/min, using A solution (5 mmol/L ammonium acetate dissolved in 0.1% acetic acid) and B solution (acetonitrile-THF=1 : 3), 90%A (0 min)-->60%A (15 min)-->const. (2 min). Mass spectral acquisition was performed in the positive mode and the analogues were targeted using multiple reaction monitoring (MRM) with electrospray ionization (ESI). The recoveries of aconitine analogues were 93-99% from Aconitum plants. The detection limits of AC, HA, MA and JA were 0.4, 0.4, 0.3 and 0.5 ng/g, respectively. The aconitine analogues, except JA, were detected in food that caused food poisoning at the level of 2.6-29.7 microg/g. These results indicate that the developed method is suitable for the determination of aconitine analogues in Aconitum plants and foods that cause food poisoning.     

LIMITATIONS OF LINEAR ELASTIC FRACTURE MECHANICS IN RESPECT OF SMALL FATIGUE CRACKS AND MICROSTRUCTURE

The growth characteristics of small fatigue cracks were investigated under rotary bending in a low alloy steel prepared with two prior austenite grain sizes of 15 μm (fine grain) and 91 μm (coarse grain). The influence of grain boundaries on crack growth rate and the crack aspect ratio was examined, and the critical crack length above which linear elastic fracture mechanics (LEFM) is applicable was evaluated for a growing small crack. When the surface crack length is shorter than three grain diameters (3d), crack growth rates decrease near the grain boundaries. Aspect ratios are also affected by the microstructure and thus vary widely. Cracks longer than 3d are not influenced by the microstructure, but they grow faster than would be expected based on LEFM until their lengths reach 3d+ 150 μm. This behaviour may be attributed to the difference in crack closure between small cracks and large cracks. If the contribution of crack closure to the growth of small cracks can be established experimentally or analytically, the critical crack length above which LEFM is applicable would be 3d. However, because it is difficult to evaluate crack closure, 3d+ 150 μm is considered to be the critical crack length for engineering applications.     

Nanoporous metal/oxide hybrid electrodes for electrochemical supercapacitors

Electrochemical supercapacitors can deliver high levels of electrical power and offer long operating lifetimes, but their energy storage density is too low for many important applications. Pseudocapacitive transition-metal oxides such as MnO(2) could be used to make electrodes in such supercapacitors, because they are predicted to have a high capacitance for storing electrical charge while also being inexpensive and not harmful to the environment. However, the poor conductivity of MnO(2) (10(-5)-10(-6) S cm(-1)) limits the charge/discharge rate for high-power applications. Here, we show that hybrid structures made of nanoporous gold and nanocrystalline MnO(2) have enhanced conductivity, resulting in a specific capacitance of the constituent MnO(2) (~1,145 F g(-1)) that is close to the theoretical value. The nanoporous gold allows electron transport through the MnO(2), and facilitates fast ion diffusion between the MnO(2) and the electrolytes while also acting as a double-layer capacitor. The high specific capacitances and charge/discharge rates offered by such hybrid structures make them promising candidates as electrodes in supercapacitors, combining high-energy storage densities with high levels of power delivery.     

THE GROWTH OF SMALL FATIGUE CRACKS IN A LOW CARBON STEEL; THE EFFECT OF MICROSTRUCTURE AND LIMITATIONS OF LINEAR ELASTIC FRACTURE MECHANICS

The growth characteristics of small fatigue cracks were studied under rotary bending in a low carbon steel prepared with two ferrite grain sizes of 24 and 84 μm, and were compared with the growth characteristics of large through cracks in fracture mechanics type specimens. The effect of microstructure on crack growth rates and the interaction in growth behaviour between two neighboring small cracks were examined experimentally, and also the critical crack lengths above which linear elastic fracture mechanics (LEFM) is applicable were evaluated for small crack growth and for fatigue crack thresholds. It is found that small cracks grow much faster than large ones and also show growth rate perturbations due to grain boundaries. It is indicated that the critical crack lengths for fatigue crack thresholds are significantly shorter than those for small crack growth.     

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.     

HTRA1 Regulates Subclinical Inflammation and Activates Proangiogenic Response in the Retina and Choroid

High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg—33% (20/60) and wild-type (WT)—7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.     

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κβ ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.